Like many functional groups or guilds of microorganisms, the group of ammonia-oxidizing bacteria (AOB) consists of a number of physiologically different species or lineages. These physiological differences suggest niche differentiation among these bacteria depending on the environmental conditions. Species of AOB might be adapted to different zones in the flooding gradient of a tidal marsh. This issue has been studied by sampling sediments from different sites and depths within a tidal freshwater marsh along the river Scheldt near the village of Appels in Belgium. Samples were taken in February, April, July and October 1998. Communities of AOB in the sediment were analysed on the basis of the 16S rRNA gene by application of polymerase chain reaction in combination with denaturing gradient gel electrophoresis (DGGE). In addition, moisture content and concentrations of ammonium and nitrate were determined as well as the potential ammonia-oxidizing activities. Six different DGGE bands belonging to the β-subclass of the Proteobacteria were observed across the marsh. The community composition of AOB was determined by the elevation in the flooding gradient as well as by the sampling depth. The presence of plants was less important for the community composition of AOB. DGGE bands affiliated with the Nitrosospira lineage were mostly found in the upper part of the marsh and in the deeper layers of the sediment. Two of the three DGGE bands related to the Nitrosomonas oligotropha lineage were more broadly distributed over the marsh, but were predominantly found in the upper layers of the sediment. Members of the environmental Nitrosomonas lineage 5 were predominantly detected in the deeper layers in the lower parts of the marsh. Potential driving factors for niche differentiation are discussed.